Strains of lactobacillus helveticus for forming exclusively L(+) lactic acid in milk

ABSTRACT

Strains of L. helveticus which form exclusively L(+) lactic acid in milk, particularly deposited strains CNCM I-1154, CNCM I-1155 and CNCM I-1156. The strains are employed to prepare an acidified milk product.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of application Ser. No.07/965, 202 which was filed Oct. 23, 1992, now U.S. Pat. No. 5,972,393.

BACKGROUND OF THE INVENTION

This invention relates to a process for the production of a dehydratedacidified milk and to the milk obtained by this process.

It is known that an acidified milk, such as a yogurt, for example in theform of a gel or beverage, can have a beneficial effect on the health.

In this connection, French Patent Application Publication No. 2 506 129(EVOG) recalls the fact that, in the traditional combinations ofmicroorganisms used to make yogurt, Lactobacillus bulgaricus isdistinguished in particular by the formation of flavour, rapidacidification and the formation of lactic acid D(-) while Streptococcusthermophilus is distinguished in particular by the formation of aconsistent gel, slow acidification and the formation of lactic acidL(+). To produce an analogous consistent gel, this patent proposes theBifidobacterium longum strain DSM 2054 which is distinguished by theformation of lactic acid L(+) and which was selected for its exceptionalacidifying power.

British Complete Patent Specification No. 1,512,890 (YAKULT HONSHA K.K.)describes the production of an acidified milk beverage by fermentationwith a combination of a strain of Lactobacillus helveticus capable ofrapidly producing lactic acid and a strain of Lactobacillus casei whichproduces very little acid, the strains being selected so that theircombination results in rapid acidification without producing anydiacetyl or other secondary product which would provide the end productwith an undesirable flavour.

U.S. Pat. No. 4,399,220 (Smiley) recalls that Lactobacillus helveticus,more particularly L. helveticus subspecies jugurti, is traditionallyused in the production of hard cheeses. This US patent proposes aprocess for the production of a culture of such a microorganism of whichthe acidifying power remains particularly stable.

SUMMARY OF THE INVENTION

The problem addressed by the present invention was to provide a processfor the production of a dehydrated acidified milk which could be carriedout on an industrial scale, which would enable relatively low pH valuesto be reached without causing any separation of proteins and which givesa product distinguished in particular by its good keeping properties andits favourable effects on the health of the consumers for which it isintended, particularly young children and babies.

To this end, in the process for the production of a dehydrated acidifiedmilk according to the invention,

an aqueous solution or emulsion is prepared with a dry matter content of10 to 40% and a composition similar to that of milk,

the solution is fermented to a pH of 4.0 to 5.0 with a combination of atleast one strain of Streptococcus thermophilus and a strain ofLactobacillus helveticus exclusively forming lactic acid L(+) and

the solution is dried to a water content below 3% by weight.

It has been found that this process can be effectively carried out on anindustrial scale without any risk of protein separation and the problemsit causes in spite of the relatively low pH value reached during thefermentation step. Similarly, the product obtained by this process hasgood keeping properties, it does not undergo any changes in itsorganoleptic qualities, particularly after storage for 12 to 24 monthsat a temperature of 20 to 27° C. in an inert gas atmosphere, for exampleof CO₂ and/or N₂, in hermetically sealed lacquered tin cans, and iseffectively capable of exerting favourable effects on the health of theconsumers for which it is intended.

In accordance with employing a strain of L. helveticus which exclusivelyforms L(+) lactic acid for carrying out the process of the presentinvention, three strains were lodged by way of example under theBudapest Treaty on Oct. 15, 1991, in the collection National de Culturesde Microorganismes (CNCM), Institut Pasteur, 28 rue de Dr. Roux, 75724Paris, Cedex 15, France, where they were given the numbers CNCM I-1154,CNCM I-1155 and CNCM I-1156.

DESCRIPTION OF THE INVENTION

The process according to the invention may be carried out using anystarting materials capable of combining the components which, together,form a composition similar to that of a milk, such as cow's milk orhuman milk.

A first preferred embodiment of the process according to the inventioncomprises preparing an aqueous solution having a dry matter content of15 to 40% by weight and a composition similar to that of a whole orskimmed cow's milk, for example optionally containing an added lacticand/or vegetable fat, such as butter oil, corn oil and/or coconut oil,and carbohydrates such as, for example, maltodextrin, sucrose, starchand/or lactose. To this end, it is possible to reconstitute apowder-form cow's milk or to concentrate a cowls milk to the desired drymatter content and optionally to add the fat and/or carbohydratesmentioned above.

A solution of the type in question may contain proteins consisting of 75to 82% by weight casein and 18 to 25% by weight whey proteins.

The solution is preferably spray-dried after fermentation to a pH valueof 4.0 to 5.0.

A second preferred embodiment of the process according to the inventioncomprises preparing an aqueous solution having a dry matter content of10 to 40% by weight and a composition adapted to the needs of babies. Tothis end, it is possible to combine the components such as a firstfraction of mineral salts, acid casein or potassium caseinate, serumproteins, cream and/or corn oil and milk powder, more particularly inratios designed to simulate the composition of human milk, optionally toadd carbohydrates, such as for example maltodextrin, sucrose, starchand/or lactose, and to reconstitute the whole to the desired dry mattercontent, for example by mixing with softened water. The solution may behomogenized at this stage, more particularly in one or two steps, forexample under a pressure of 50 to 200 bar.

The solution thus formed may contain proteins consisting of 40 to 82% byweight casein and 18 to 60% by weight whey proteins.

After the solution has been fermented to a pH value of 4.0 to 5.0, it ispreferably concentrated to a dry matter content of 40 to 55% by weightby evaporation in vacuo and supplemented with vitamins and, optionally,a second fraction of mineral salts and then spray-dried. This optionalsecond fraction of mineral salts may comprise, in particular, salts ofCa and Mg which are preferably added after evaporation and either beforeor even during or after drying.

It has been found that it can also be of advantage to add lecithineither during the initial mixing or after evaporation and before drying.Finally, optional carbohydrates may also be added after drying ratherthan during the initial mixing, particularly in hot climates where thereis a risk of problems caused by sticking in the drying nozzle.

In each preferred embodiment of the process according to the invention,the solution is fermented by inoculation with, preferably, 1 to 5% byvolume of a culture or mixture of cultures containing 10⁷ to 10⁹ germsof at least one S. thermophilus and 10⁷ -10⁹ germs of L. helveticus perml and incubation for 2 to 15 h at a temperature of 37 to 45° C.

This fermentation may be carried out discontinuously, i.e. in batches,or continuously. Thus, in one advantageous embodiment of the processaccording to the invention which is particularly suitable forapplication on an industrial scale, fermentation is carried outcontinuously in two successive steps. In the first of these steps,incubation may be carried out for 0.5 to 5 h to pH 5.0 to 6.0 andpreferably to pH 5.1 to 5.7 at a temperature of 37 to 45° C. In thesecond of these steps, incubation may be carried out for 1 to 10 h to pH4.0 to 5.0 and preferably to pH 4.5 to 4.8 at a temperature of 37 to 45°C.

These fermentation steps may be carried out in two successive fermenterson a continuous production line, the volume of solution treated in eachfermenter being proportional to the desired residence time for example.

This method of operation lends itself particularly well to optimal workof each of the two microorganisms forming the combination in symbiosis,the first fermentation step favouring, in particular, the work of the S.thermophilus strain(s) and the second step favouring, in particular, thework of the L. helveticus strain.

In each embodiment of the process according to the invention, the S.thermophilus strain(s) may be selected, for example, from the strainsmarketed for the production of yogurts or isolated from yogurts. The L.helveticus strain may be selected for its ability to exclusively formlactic acid L(+), for example from the strains marketed for theproduction of cheese or acidifed milk or isolated from cheeses oracidified milks, and details of the morphology and properties of strainsCNCM I-1154, CNCM I-1155 and CNCM I-1156 are shown below.

Morphology

Examined under a microscope, these strains are in the form of relativelylong and thin rodlets.

They are microaerophilic gram-positive bacteria which do not formspores.

Metabolism

These bacteria produce homolactic fermentation with formation of lactateas the end product.

Various tests with each of them give the following results: catalaseactivity (-), production of carbon dioxide (-), coagulation of milk (+),hydrolysis of arginine (-)

They ferment sugars as shown in the tabular listing set forth herein.

    ______________________________________                                                 CNCM       CNCM    CNCM                                                       I-1154     I-1155  I-1156                                            ______________________________________                                        L-Arabinose                                                                              -            -       -                                             D-Xylose   -            -       -                                             D-Glucose  +            +       +                                             D-Mannose  +            -       +                                             D-Fructose +            -       -                                             Galactose  +            +       +                                             Lactose    +            +       +                                             Maltose    -            -       -                                             Sucrose    -            -       -                                             Trehalose  -            -       -                                             Raffinose  -            -       -                                             Salicine   -            -       -                                             Cellobiose -            -       -                                             D-Sorbitol -            -       -                                             D-Mannitol -            -       -                                             Inositol   -            -       -                                             Cellobiose -            -       -                                             Glycerine  -            -       -                                             Starch     -            -       -                                             ______________________________________                                    

Growth medium

These strains may be grown on MRS medium or reconstituted skimmed milkcontaining 10% dry matter. The optimal temperature is 40-45° C. The pHafter 24 h in reconstituted skimmed milk is approx. 3.40.

Feature

These three strains exclusively (>99%) produce lactic acid L(+).

Products Obtained

The present invention also relates to the product obtained by theprocess according to the invention. This product is intended inparticular for young children and babies on whose health it can exert abeneficial effect, particularly in preventing diarrhoea, by comparisonwith comparable traditional products.

In one particular embodiment of the product, a lyophilized culture ofBifidobacterium may also be added.

EXAMPLE

The following Examples are intended to illustrate the process and theproduct according to the invention. Various tests are also described toillustrate the qualities of the product. In the Examples and tests,percentages are by weight unless otherwise indicated.

Example 1

Corn oil, sucrose, maltodextrin and starch are added to a whole cow'smilk so that the solution obtained has the following composition (in %by weight, based on dry matter):

    ______________________________________                                        Milk fats            18.2                                                     Corn oil              4.6                                                     Milk proteins        14.5                                                     (including approx. 80% casein and                                             approx. 20% whey proteins)                                                    Lactose              21.2                                                     Sucrose              15.5                                                     Maltodextrin         16.5                                                     Starch                6.2                                                     Minerals              3.2                                                     ______________________________________                                    

This solution is concentrated to 33% dry matter by evaporation in vacuo.

The concentrated solution is inoculated with 5% by volume of a culturecontaining per ml approx. 10⁸ germs of a commercial Streptococcusthermophilus and approx. 10⁸ germs of the Lactobacillus helveticusstrain CNCM I-1154. The concentrated solution may be fermented for about12.5 h at 43° C. to a pH value of 4.2 to 4.3. The fermented solution isspray-dried.

A dehydrated acidified milk, i.e. an acidified milk powder, intended inparticular for young children is obtained and may be reconstituted withwater in a quantity of approx. 140 g powder to approx. 900 ml water.This powder has a residual water content of approximately 2% and keepswithout changing for at least 12 months at 27° C. in hermetically sealedlacquered tin cans.

Example 2

The procedure is as described in Example 1 except that the concentratedsolution is inoculated with the Lactobacillus helveticus strain CNCMI-1155 instead of the strain CNCM I-1154.

The dehydrated acidified milk obtained is comparable in its propertieswith the product obtained in Example 1.

Example 3

The procedure is as described in Example 1, except that the concentratedsolution is inoculated with the Lactobacillus helveticus strain CNCMI-1156 instead of strain CNCM I-1154.

The dehydrated acidified milk obtained is comparable in its propertieswith the product obtained in Example 1.

Test 1

The dehydrated acidified milk obtained in Example 1 is subjected to amicrobial proliferation test in a feeding bottle.

To this end, the dehydrated acidified milk is reconstituted with waterin a quantity of 139 g powder to 900 ml water and is inoculated withvarious pathogenic microorganisms.

For comparison, a reconstituted non-acidified dehydrated milk adapted inits composition to the needs of babies is inoculated with the samemicroorganisms.

The pathogenic microorganisms used are Salmonella typhimurium,Escherichia coli, Staphylococcus aureus and Bacillus cereus. They areinoculated in a quantity of 10⁴ -10⁵ german per ml reconstituted milk.The respective populations are counted after incubation for 8 h at 37°C. The results obtained are set out in Table 1 below:

                  TABLE 1                                                         ______________________________________                                        Microbial proliferation test in a feeding bottle                              (initial inoculation with 10.sup.4 -10.sup.5 germs/ml - incubation            for 8 h at 37° C.).                                                    Product tested                                                                          S. typhimurium                                                                           E. coli  St. aureus                                                                           B. cereus                                ______________________________________                                        Non-acidified                                                                           4.2 • 10.sup.7                                                                     9.1 • 10.sup.7                                                                   8.0 • 10.sup.6                                                                 8.0 • 10.sup.6                     comparison                                                                    sample                                                                        Acidified de-                                                                           <10        <10      2.2 • 10.sup.3                                                                 2.3 • 10.sup.4                     hydrated milk                                                                 acc. to Ex. 1                                                                 ______________________________________                                    

It can be seen from Table 1 that the acidified dehydrated milk obtainedin Example 1 completely inhibits the growth of Staph. aureus and B.Cereus and even inactivates S. typhimurium and E. coli.

Example 4

A first fraction of mineral salts, potassium caseinate, demineralizedwhey proteins, cream, skimmed milk powder, maltodextrin and softenedwater are mixed to obtain a solution having a dry matter content of 20%by weight and the following composition (in % by weight, based on drymatter):

    ______________________________________                                        Milk fats             19.4                                                    Corn oil               4.8                                                    (subsequently added hot)                                                      Lecithin               0.5                                                    (subsequently added hot)                                                      Milk proteins         12.9                                                    (50% casein, 50% whey proteins)                                               Lactose               45.4                                                    Maltodextrin          15.0                                                    Minerals               2.0                                                    (including Ca and Mg added before,                                            during or after drying)                                                       ______________________________________                                    

After preheating to 80° C., the corn oil is added. The solution isheat-treated by injection of steam at 125° C. for 5 s. The solution isthen homogenized under a pressure of 75 bar at 43° C. The homogenizedsolution or emulsion is then inoculated with 2% by volume of a culturecontaining per ml approx. 108 germs of a commercial Streptococcusthermophilus and approx. 108 germs of the Lactobacillus helveticusstrain CNCM I-1154.

The solution is fermented for 4.5 h at 43° C. to a pH value of 4.2 to4.3 and is then cooled to 10° C.

The solution is then preheated to 75° C. and concentrated by evaporationto 48% dry matter. Lecithin is added. A second fraction of mineralsalts, namely salts of Ca and Mg, is then added and the solution isspray-dried.

A dehydrated acidified milk, i.e. an acidified milk powder, intended inparticular for babies is obtained and may be reconstituted with water ina quantity of 135 g powder to approx. 900 ml water. The powder has aresidual water content of approx. 2% and keeps without changing for atleast 12 months at 27° C. in hermetically sealed lacquered tin cans.

Example 5

The procedure is as described in Example 4, except that the solution oremulsion is inoculated with the Lactobacillus helveticus strain CNCMI-1155 instead of strain CNCM I-1154.

The dehydrated acidified milk obtained is comparable in its properteswith the product obtained in Example 4.

Example 6

The procedure is as described in Example 4, except that the solution oremulsion is inoculated with the Lactobacillus helveticus strain CNCMI-1156 instead of strain CNCM I-1154.

The dehydrated acidified milk obtained is comparable in its properteswith the product obtained in Example 4.

Test 2

The dehydrated acidified milk obtained in Example 4 is subjected to amicrobial proliferation test in a feeding bottle.

To this end, the dehydrated acidified milk is reconstituted with waterin a quantity of 134 g powder to 900 ml water and inoculated withvarious pathogenic microorganisms in the same way as described above intest 1.

Table 2 below shows the results obtained after incubation for 8 h at 37°C. against the results obtained for comparison with a sample ofreconstituted dehydrated milk similar in composition, but not acidified.

                  TABLE 2                                                         ______________________________________                                        Microbial proliferation test in a feeding bottle                              (initial inoculation with 10.sup.4 -10.sup.5 germs/ml - incubation            for 8 h at 37° C.).                                                    Product tested                                                                          S. typhimurium                                                                           E. coli  St. aureus                                                                           B. cereus                                ______________________________________                                        Non-acidified                                                                           4.2 • 10.sup.7                                                                     9.1 • 10.sup.7                                                                   8.0 • 10.sup.6                                                                 8.0 • 10.sup.6                     comparison                                                                    sample                                                                        Acidified de-                                                                           10         10       1.3 • 10.sup.4                                                                 1.9 • 10.sup.4                     hydrated milk                                                                 acc. to Ex. 4                                                                 ______________________________________                                    

Accordingly, it can be seen from Table 2 that the acidified dehydratedmilk obtained in Example 4 completely inhibits the growth of these fourpathogenic microorganisms and even inactivates S. typhimurium and E.coli.

Test 3

An acceptance study was carried out on the acidified dehydrated milkobtained in Example 4.

The study involves 25 babies during the first three months of theirlives. The development of their size and weight is followed and theregurgitations, the frequency of stools and their consistency arerecorded.

Growth curves established from the results of this study are situatedright in the middle of the ranges considered as normal in Europe andshow a slightly more pronounced slope than a typical average curve.

The results of monitoring of the regurgitations and stools are set outin Table 3 below which shows the average quantities of milk consumed perday in ml per kg baby (values in brackets) and the percentage of babiesin which the number of regurgitations is below 2 per day and of whichthe frequency and consistency of the daily stools correspond to thenumbers and descriptions indicated.

                  TABLE 3                                                         ______________________________________                                        Acceptance test of the acidified dehydrated milk                              according to Example 4 in babies                                                        First month                                                                            Second month                                                                             Third month                                     ______________________________________                                        Consumption of                                                                            175        160        142                                         milk (ml/kg (120-243)  (107-203)  (85-181)                                    Regurgitation <2                                                                           86         94         93                                         Frequency of stools                                                           <1           4          10         18                                         1-3          83         80         70                                         <4           13         10         12                                         Consistency of stools                                                         firm         63         54         50                                         soft         33         32         35                                         liquid       4          14         15                                         ______________________________________                                    

The results set forth in Table 3 reflect an excellent tolerance of thedehydrated acidified milk by the babies and a relatively low tendencytowards regurgitation.

Example 7

A first fraction of mineral salts, potassium caseinate, demineralizedwhey proteins, cream, skimmed milk powder, maltodextrin and softenedwater are continuously mixed to obtain 4000 liters per hour of asolution having a dry matter content of 20% by weight and the followingcomposition (in % by weight, based on dry matter):

    ______________________________________                                        Milk fats             19.2                                                    Corn oil               5.3                                                    (subsequently added hot)                                                      Lecithin               0.5                                                    (subsequently added hot)                                                      Milk proteins         12.8                                                    (50% casein, 50% whey proteins)                                               Lactose               45.7                                                    Maltodextrin          14.6                                                    Minerals               1.9                                                    (including Ca and Mg added before,                                            during or after drying)                                                       ______________________________________                                    

The solution is continuously preheated to 80° C. in a plate-type heatexchanger. Corn oil is added. The solution is then heat-treated byinjection of steam at 125° C. for 5 s. The solution is homogenized intwo steps at 43° C., the first under a pressure of 150 bar and thesecond under a pressure of 50 bar. The homogenized solution or emulsionis inoculated with 1% by volume of a culture containing per ml approx.5.108 germs of two commercial Streptococcus thermophilus strains and 3%by volume of a culture containing per ml approx. 2.10⁸ germs of theLactobacillus helveticus strain CNCM I-1154.

The solution is fermented first for about 1 h at 42° C. to pH 5.5 in a4000 l fermenter and then for about 1.5 h at 42° C. to pH 4.7 in a 6000l fermenter. The solution is preheated to 75° C. in a plate-type heatexchanger. It is then concentrated by evaporation to a dry mattercontent of 50% in a three-stage falling-film evaporator. The lecithin isthen added. A second fraction of mineral salts, namely salts of Ca andMg, is added and the solution is then spray-dried.

A dehydrated acidified milk, i.e. an acidified milk powder, intended inparticular for babies is continuously obtained and may be reconstitutedwith water in a quantity of approx. 134 g powder to approx. 900 mlwater. The powder has a residual water content of approx. 2% and keepswithout changing for at least 12 months at 27° C. in hermetically sealedlacquered tin cans.

Example 8

The procedure is as described in Example 7, except that the solution oremulsion is inoculated with the Lactobacillus helveticus strain CNCMI-1155 instead of the strain CNCM I-1154 and the second fraction ofmineral salts is not added until after spray drying.

The dehydrated acidified milk obtained is comparable in its propertieswith the product obtained in Example 7.

Example 9

The procedure is as described in Example 7 except that the solution oremulsion is inoculated with the Lactobacillus helveticus strain CNCMI-1156 instead of the strain CNCM I-1154. The dehydrated acidified milkobtained is comparable in its properties with the product obtained inExample 7.

Example 10

A lyophilized commercially available Bifidobacterium is added to theproduct obtained in any of Examples 4 to 9 in such a quantity that thepowder has a content of approx. 5.10⁷ Bifidobacterium germs per g.

What is claimed is:
 1. A culture of isolated bacteria strain L.helveticus CNCM I-1154.
 2. A culture of isolated bacteria strain L.helveticus CNCM I-1155.
 3. A culture of isolated bacteria strain L.helveticus CNCM I-1156.
 4. A culture comprising bacteria selected fromthe group consisting of isolated bacteria strains L. helveticus CNCMI-1154, CNCM I-1155 and CNCM I-1156 and comprising an S. thermophilusbacteria strain for producing yogurt.